Methods of BH3 profiling

ABSTRACT

In various aspects the invention provides methods of predicting sensitivity of a cancer cell to a therapeutic agent by contacting a test cell population BH3 domain peptide; measuring the amount of BH3 domain peptide induced mitochondrial outer membrane permeabilization in the test cell population; and comparing the amount of BH3 domain peptide induced mitochondrial outer membrane permeabilization in the test cell population to a control cell population that has not been contacted with the therapeutic agent. An increase in mitochondrial membrane permeabilization in the test cell population compared to the control cell population indicates the cell is sensitive to the therapeutic agent.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofInternational Application No. PCT/US2014/056284, filed Sep. 18, 2014,and entitled “METHODS OF BH3 PROFILING,” which claims the benefit under35 U.S.C. § 119(e) of U.S. provisional application Ser. No. 61/879,869,filed Sep. 19, 2013, the contents of which are incorporated by referenceherein in their entirety.

FIELD OF THE INVENTION

The invention relates to generally to improved methods of determiningcellular chemosensitivity by determining the pattern of sensitively of acell to a panel of BH3 domain peptides.

BACKGROUND OF THE INVENTION

Programmed cell death, referred to as apoptosis, plays an indispensablerole in the development and maintenance of tissue homeostasis within allmulticellular organisms (Raff, Nature 356: 397-400, 1992). Genetic andmolecular analysis from nematodes to humans has indicated that theapoptotic pathway of cellular suicide is highly conserved (Hengartnerand Horvitz, Cell 76: 1107-1114, 1994). In addition to being essentialfor normal development and maintenance, apoptosis is important in thedefense against viral infection and in preventing the emergence ofcancer.

Diverse intrinsic death signals emanating from multiple subcellularlocales all induce the release of cytochrome c from mitochondria toactivate Apaf-1 and result in effector caspase activation. Proteins inthe BCL-2 family are major regulators of the commitment to programmedcell death as well as executioners of death signals at themitochondrion. Members of this family include both pro- andanti-apoptotic proteins and share homology in up to four conservedregions termed BCL-2 homology (BH) 1-4 domains (Adams and Cory, 1998).The family can be divided into three main sub-classes. Theanti-apoptotic proteins, which include BCL-2 and BCL-X_(L), are all“multidomain,” sharing homology throughout all four BH domains. However,the pro-apoptotic proteins can be further subdivided and includemultidomain proteins, such as BAX and BAK, which possess sequencehomology in BH1-3 domains. The more distantly related “BH3-only”proteins are to date all pro-apoptotic and share sequence homologywithin the amphipathic α-helical BH3 region, which is required for theirapoptotic function (Chittenden et al., 1995; O'Connor et al., 1998; Wanget al., 1996; Zha et al., 1997).

Multidomain pro-apoptotic proteins such as BAX and BAK upon receipt ofdeath signals participate in executing mitochondrial dysfunction. Inviable cells, these proteins exist as monomers. In response to a varietyof death stimuli, however, inactive BAX, which is located in the cytosolor loosely attached to membranes, inserts deeply into the outermitochondrial membrane as a homo-oligomerized multimer (Eskes et al.,2000; Gross et al., 1998; Wolter et al., 1997). Inactive BAK resides atthe mitochondrion where it also undergoes an allosteric conformationalchange in response to death signals, which includes homo-oligomerization(Griffiths et al., 1999; Wei et al., 2000). Cells deficient in both BAXand BAK are resistant to a wide variety of death stimuli that emanatefrom multiple locations within the cell (Wei et al., 2001).

The BH3-only molecules constitute the third subset of this family andinclude BID, NOXA, PUMA, BIK, BIM and BAD (Kelekar and Thompson, 1998).These proteins share sequence homology only in the amphipathic α-helicalBH3 region which mutation analysis indicated is required inpro-apoptotic members for their death activity. Moreover, the BH3-onlyproteins require this domain to demonstrate binding to “multidomain”BCL-2 family members. Multiple binding assays, including yeasttwo-hybrid, co-immunoprecipitation from detergent solubilized celllysates and in-vitro pull down experiments indicate that individualBH3-only molecules display some selectivity for multidomain BCL-2members (Boyd et al., 1995; O'Connor et al., 1998; Oda et al., 2000;Wang et al., 1996; Yang et al., 1995). The BID protein bindspro-apoptotic BAX and BAK as well as anti-apoptotic BCL-2 and BCL-X_(L)(Wang et al., 1996; Wei et al., 2000). In contrast, BAD, and NOXA asintact molecules display preferential binding to anti-apoptotic members(Boyd et al., 1995; O'Connor et al., 1998; Oda et al., 2000; Yang etal., 1995)

SUMMARY OF THE INVENTION

In various aspects the invention provides methods of predictingsensitivity of a cancer cell to a therapeutic agent by contacting a testcell population with a BH3 domain peptide; measuring the amount of BH3domain peptide induced mitochondrial outer membrane permeabilization inthe test cell population; and comparing the amount of BH3 domain peptideinduced mitochondrial outer membrane permeabilization in the test cellpopulation to a control cell population that has not been contacted withthe therapeutic agent. An increase in mitochondrial membranepermeabilization in the test cell population compared to the controlcell population indicates the cell is sensitive to the therapeuticagent. Optionally, the cell is permeabilized prior to contacting withsaid BH3 domain peptide.

Mitochondrial outer membrane permeabilization is determined for exampleby measuring i) the emission of a potentiometric or radiometric dye orii) the release of molecules from the mitochondrial inter-membranespace.

In some embodiments the permeabilized cells are contacted with apotentiometric dye such as JC-1 or dihydrorhodamine 123. In otherembodiments, the permeabilized cell is contacted with an antibody forcytochrome C or SMAC/Diablo, Omi, adenylate kinase-2 or apoptosisinducing factor.

Optionally, the method further includes contacting said permeabilizedcell with an antibody for an intracellular or extracellular marker.

In some aspects the cell population is fixed prior to measuringmitochondrial outer membrane permeabilization. For example, the cellpopulation is fixed on a solid surface.

A BH3 domain peptide is derived from the BH3 domain of a BID, a BIM, aBAD, a BIK, a NOXA, a PUMA a BMF, or a HRK polypeptide.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of bar graphs demonstrating that iBH3 can reproducethe profile of individual subpopulations with mixed populations. Samplesprofiled individually (unmixed as shown in FIG. 1A) or as a complexmixture (mixed as shown in FIG. 1B) produce the same profile.

FIG. 2 is a series of panels showing how iBH3 defines cell populationsand measures cellular response to profiling. Representative FACS data(FIG. 2A and FIG. 2B) demonstrate the isolation of subpopulations withinthe mixed sample in FIG. 1.

FIG. 3 is a series of fluorescent microscopy images that show the lossof cytochrome c in response to peptide treatment measured by microscopy.Cells are located by DAPI staining of their nuclei, mitochondria arelocated by staining of a mitochondrial marker (MnSOD) adjacent tonuclei, and cytochrome c staining is correlated with regions ofmitochondrial marker staining. An inert control peptide shows cytochromec staining in regions of MnSOD staining while BIM peptide causes almosttotal loss of cytochrome c from all regions of MnSOD staining.

FIG. 4 is a series of bar graphs showing correlation of miBH3 profileswith known profiles. The miBH3 profile of the SuDHL4 cell line (FIG. 4A)shows loss of correlation between cytochrome c and MnSOD channels inresponse to BH3 peptides. Release of cytochrome c and loss ofcorrelation for BIM, BAD, PUMA, and BMF peptides match the loss ofcytochrome c measured by other BH3 profiling methods shown in FIG. 4B.

FIG. 5 is a graph showing that pre-made frozen plates perform the sameas freshly prepared plates. Responsive cells (MDA-MB-231) showcomparable response to a peptide treatment (BAD) in both frozen andfreshly prepared plates. Non-responsive cells (SuDHL10) are used to testfor non-specific noise, and frozen plates produce a response equivalentto freshly prepared plates.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part by the discovery of improvedmethods of measuring mitochondrial outer membrane permeabilization.These improved methods are useful in BH3 Profiling as described inUS2008/0199890, the contents of which are incorporated by reference inits entirety.

BH3 Profiling

In various methods, sensitivity of a cell to an agent is determined.Cell sensitivity is determined by contacting a cell or cellularcomponent (e.g., mitochondria) with a BH3 domain peptide. A cell issensitive to an agent if apoptosis is detected. Alternatively, cellsensitivity is determined by providing a test BH3 profile of the celland comparing the profile to a cancer cell BH3 profile. A similarity ofthe test profile and the control profile indicates that the cell issensitive to an agent. A BH3 profile is a pattern of sensitivity to BH3peptides of the cell. Sensitivity is indicated by apoptosis. A cancercell BH3 profile is a pattern of sensitivity to BH3 peptides in a cancercell whose responsiveness or lack thereof to a particular agent isknown. Optionally, the test BH3 profile is compared to more than onecancer cell BH3 profile. Thus, by comparing the test BH3 profile to thecontrol BH3 profile sensitivity to an agent is determined.

The cell or cellular component is a cancer cell or a cell that issuspected of being cancerous. The cell is permeabilized to permit theBH3 peptides access to the mitochondria. Cells are permeabilized bymethods known in the art, for example, the cells are permeabilized bycontacting the cell with digitonin.

After the cells are permeabilized the cells are treated with the BH3peptides or test agents. After the cell is treated, mitochondrial outermembrane permeabilization is measured. Outer membrane permeabilizationis measured by a number of methods, for example, outer membranepermeabilization by loss of mitochondrial membrane potential. Loss ofmitochondrial membrane potential is measured for example by treating thecells with a potentiometric or radiometric dye.

Alternatively, outer membrane permeabilization is determined bymeasuring the release of molecules from the mitochondrial inter-membranespace. Examples of molecules that can be measured include cytochrome cand SMAC/Diablo, Omi, adenylate kinase-2 or apoptosis inducing factor(AIF). Optionally, the cells are fixed prior to measuring outer membranepermeabilization. Cells are fixed by methods known in the art such as byusing an aldehyde such as formaldehyde.

Mitochondrial outer membrane permeabilization can be measured at thesingle cell level or multi-cell level or across the entire population ofcells. Additionally, some of the methods disclosed herein allow forsubpopulations of cells to be assayed.

Examples of potentiometric dyes include green-fluorescent JC-1 probe(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanineiodide) or dihydrorhodamine 123.

JC-1 exists as a monomer at low membrane concentrations). However, JC-1accumulates in the mitochondrial matrix under conditions of highermitochondrial potentials. At these higher concentrations, JC-1 formsred-fluorescent “J-aggregates”. As a monomer the dye has anabsorption/emission maxima of 527 nm while at high membrane potentialthe emission maximum is 590 nm. Thus, ratio measurements of the emissionof this cyanine dye can be used as a sensitive measure of mitochondrialmembrane potential. The dye allows for a dual measurement of dyeconcentration that does not require the measurement of a nuclear orcytoplasmic reference values. Studies using isolated mitochondria haveshown that the 527 nm emission from monomeric JC-1 increases almostlinearly with membrane M potentials ranging from 46 to 182 mV, whereasthe 590 nm J-aggregate emission is less sensitive to M values lessnegative than 140 my and is strongly sensitive to potential values inthe range of 140 to 182 mV (Di Lisa et al., 1995) Optical filtersdesigned for fluorescein and tetramethylrhodamine can be used toseparately visualize the monomer and J-aggregate forms, respectively.Alternatively, both forms can be observed simultaneously using astandard fluorescein long pass optical filter set.

Dihydrorhodamine 123 is an uncharged, nonfluorescent agent that can beconverted by oxidation to the fluorescent laser dye rhodamine 123(R123).

Release of molecules from the mitochondrial inter-membrane space can bemeasured by methods known in the art, for example, by using antibodiesto the molecules to be measured, i.e., antibodies to cytochrome C orSMAC/Diablo. Detection can be for example, by ELISA, FACS, immunoblot,immunofluorescence, or immunohistochemistry.

In addition to measuring molecules that get released from themitochondrial space, other intracellular and extracellular markers canbe measured. This allows for the ability to discriminate betweensubpopulations of cells.

BH3 profiling can be accomplished at the single cell level byimmobilizing cells on a solid surface. Optionally the solid surface ispolyamine or poly-lysine coated. Immobilized cells are permeabilized asdescribed above. The cells are then contacted with BH3 peptides and/ortest agents. After the cells have been treated for a predeterminedperiod of time such as 45-90 minutes, the cells are fixed and furtherpermeabilized by methods known in the art. For example the cells arefixed with formaldehyde and further permeabilized with methanol orTriton X-100 (t-Octylphenoxypolyethoxyethanol). Outer membranepermeabilization is determined by intracellular staining for moleculesfrom the mitochondrial inter-membrane space and a mitochondrial marker.Examples of molecules that can be measured include cytochrome c,SMAC/Diablo, Omi, adenylate kinase-2 or apoptosis inducing factor (AIF).A mitochondrial marker includes MnSOD. Stained cells can becounterstained with nuclear stains such as DAPI. Optionally otherintracellular and extracellular markers can be measured. Analysis of thecells can be manually accomplished using a microscope or automated forexample by using software such as Cellprofiler to locate nuclei. Thecell is from a subject known to or suspected of having cancer. Thesubject is preferably a mammal. The mammal is, e.g., a human, non-humanprimate, mouse, rat, dog, cat, horse, or cow. The subject has beenpreviously diagnosed as having cancer, and possibly has alreadyundergone treatment for cancer. Alternatively, the subject has not beenpreviously diagnosed as having cancer.

The agent is a therapeutic agent such as a chemotherapeutic agent. Forexample the agent is a mimetic of sensitizer BH3 domains or anantagonist of an anti-apoptotic protein. Apoptosis, i.e., cell death isidentified by known methods. For example, characteristics of apoptosisinclude the cell shrinks, develop bubble-like blebs on their surface,have the chromatin (DNA and protein) in their nucleus degraded, and havetheir mitochondria break down with the release of cytochrome c, loss ofmitochondrial membrane potential, break into small, membrane-wrapped,fragments, or phosphatidylserine, which is normally hidden within theplasma membrane, is exposed on the surface of the cell.

The difference in the level apoptosis of a cell that has been contactedwith a BH3 peptide compared to a cell that has not been contacted with aBH3 peptide is statistically significant. By statistically significant,it is meant that the alteration is greater than what might be expectedto happen by chance alone. Statistical significance is determined bymethod known in the art. For example statistical significance isdetermined by p-value. The p-value is a measure of probability that adifference between groups during an experiment happened by chance.(P(z≥z_(observed))). For example, a p-value of 0.01 means that there isa 1 in 100 chance the result occurred by chance. The lower the p-value,the more likely it is that the difference between groups was caused bytreatment. An alteration is statistically significant if the p-value isor less than 0.05. Preferably, the p-value is 0.04, 0.03, 0.02, 0.01,0.005, 0.001 or less.

The invention also includes a profile of a pattern of mitochondrialsensitivity to BH3 sensitizer peptides taken from one or more subjectswho have cancer.

BH3 Domain Peptides

A BH3 domain peptide is less than 195 amino acids in length, e.g., lessthan or equal to 150, 100, 75, 50, 35, 25 or 15 amino acids in length.For example a BH3 peptide includes the sequence of SEQ ID NO: 1-13 shownin Table 1.

TABLE 1 AMINO ACID SEQUENCE SEQ ID NO BID EDIIRNIARHLAQVGDSMDR  1 BIMMRPEIWIAQELRRIGDEFNA  2 BID mut EDIIRNIARHAAQVGASMDR  3 BADLWAAQRYGRELRRMSDEFEGSFKGL  4 BIK MEGSDALALRLACIGDEMDV  5 NOXA AAELPPEFAAQLRKIGDKVYC  6 NOXA B PADLKDECAQLRRIGDKVNL  7 HRKSSAAQLTAARLKALGDELHQ  8 BNIP VVEGEKEVEALKKSADWVSD  9 PUMAEQWAREIGAQLRRMADDLNA 10 BMF HQAEVQIARKLQLIADQFHR 11 huBADNLWAAQRYGRELRRMSDEFVDSFK 12 K BAD mut LWAAQRYGREARRMSDEFEGSFKGL 13

A BH3 domain peptide include a peptide which includes (in whole or inpart) the sequence NH₂-XXXXXXIAXXLXXXGDXXXX-COOH (SEQ ID NO:14) orNH₂-XXXXXXXXXXLXXXXDXXXX-COOH (SEQ ID NO:15). As used herein X may beany amino acid. Alternatively, the BH3 domain peptides include at least5, 6, 7, 8, 9, 15 or more amino acids of SEQ ID NO:14 or SEQ ID NO:15.

Optionally, the BH3 domain peptide is attached to transduction domain. Atransduction domain is a compound that directs a peptide in which it ispresent to a desired cellular destination. Thus, the transduction domaincan direct the peptide across the plasma membrane, e.g., from outsidethe cell, through the plasma membrane, and into the cytoplasm.Alternatively, or in addition, the transduction domain can direct thepeptide to a desired location within the cell, e.g., the nucleus, theribosome, the ER, mitochondria, a lysosome, or peroxisome.

In some embodiments, the transduction domain is derived from a knownmembrane-translocating sequence. Alternatively, the transduction domainis a compound that is known to facilitate membrane uptake such aspolyethylene glycol, cholesterol moieties, octanoic acid and decanoicacid.

For example, the trafficking peptide may include sequences from thehuman immunodeficiency virus (HIV) 1 TAT protein. This protein isdescribed in, e.g., U.S. Pat. Nos. 5,804,604 and 5,674,980, eachincorporated herein by reference. The BH3 domain peptide is linked tosome or all of the entire 86 amino acids that make up the TAT protein.For example, a functionally effective fragment or portion of a TATprotein that has fewer than 86 amino acids, which exhibits uptake intocells can be used. See e.g., Vives et al., J. Biol. Chem.,272(25):16010-17 (1997), incorporated herein by reference in itsentirety. A TAT peptide that includes the region that mediates entry anduptake into cells can be further defined using known techniques. See,e.g., Franked et al., Proc. Natl. Acad. Sci, USA 86: 7397-7401 (1989).Other sources for translocating sequences include, e.g., VP22 (describedin, e.g., WO 97/05265; Elliott and O'Hare, Cell 88: 223-233 (1997)),Drosophila Antennapedia (Antp) homeotic transcription factor, HSV,poly-arginine, poly lysine, or non-viral proteins (Jackson et al, Proc.Natl. Acad. Sci. USA 89: 10691-10695 (1992)).

The transduction domain may be linked either to the N-terminal or theC-terminal end of BH3 domain peptide. A hinge of two proline residuesmay be added between the transduction domain and BH3 domain peptide tocreate the full fusion peptide. Optionally, the transduction domain islinked to the BH3 domain peptide in such a way that the transductiondomain is released from the BH3 domain peptide upon entry into the cellor cellular component.

The transduction domain can be a single (i.e., continuous) amino acidsequence present in the translocating protein. Alternatively it can betwo or more amino acid sequences, which are present in protein, but inthe naturally-occurring protein are separated by other amino acidsequences.

The amino acid sequence of naturally-occurring translocation protein canbe modified, for example, by addition, deletion and/or substitution ofat least one amino acid present in the naturally-occurring protein, toproduce modified protein. Modified translocation proteins with increasedor decreased stability can be produced using known techniques. In someembodiments translocation proteins or peptides include amino acidsequences that are substantially similar, although not identical, tothat of naturally-occurring protein or portions thereof. In addition,cholesterol or other lipid derivatives can be added to translocationprotein to produce a modified protein having increased membranesolubility.

The BH3 domain peptide and the transduction domain can be linked bychemical coupling in any suitable manner known in the art. Many knownchemical cross-linking methods are non-specific, i.e.; they do notdirect the point of coupling to any particular site on the transportpolypeptide or cargo macromolecule. As a result, use of non-specificcross-linking agents may attack functional sites or sterically blockactive sites, rendering the conjugated proteins biologically inactive.

One way to increasing coupling specificity is to directly chemicalcoupling to a functional group found only once or a few times in one orboth of the polypeptides to be cross-linked. For example, in manyproteins, cysteine, which is the only protein amino acid containing athiol group, occurs only a few times. Also, for example, if apolypeptide contains no lysine residues, a cross-linking reagentspecific for primary amines will be selective for the amino terminus ofthat polypeptide. Successful utilization of this approach to increasecoupling specificity requires that the polypeptide have the suitablyrare and reactive residues in areas of the molecule that may be alteredwithout loss of the molecule's biological activity.

Cysteine residues may be replaced when they occur in parts of apolypeptide sequence where their participation in a cross-linkingreaction would otherwise likely interfere with biological activity. Whena cysteine residue is replaced, it is typically desirable to minimizeresulting changes in polypeptide folding. Changes in polypeptide foldingare minimized when the replacement is chemically and sterically similarto cysteine. For these reasons, serine is preferred as a replacement forcysteine. As demonstrated in the examples below, a cysteine residue maybe introduced into a polypeptide's amino acid sequence for cross-linkingpurposes. When a cysteine residue is introduced, introduction at or nearthe amino or carboxy terminus is preferred. Conventional methods areavailable for such amino acid sequence modifications, whether thepolypeptide of interest is produced by chemical synthesis or expressionof recombinant DNA.

Coupling of the two constituents can be accomplished via a coupling orconjugating agent. There are several intermolecular cross-linkingreagents which can be utilized, See for example, Means and Feeney,CHEMICAL MODIFICATION OF PROTEINS, Holden-Day, 1974, pp. 39-43. Amongthese reagents are, for example, J-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or N, N′-(1,3-phenylene) bismaleimide (both of whichare highly specific for sulfhydryl groups and form irreversiblelinkages); N, N′-ethylene-bis-(iodoacetamide) or other such reagenthaving 6 to 11 carbon methylene bridges (which relatively specific forsulfhydryl groups); and 1,5-difluoro-2, 4-dinitrobenzene (which formsirreversible linkages with amino and tyrosine groups). Othercross-linking reagents useful for this purpose include:p,p′-difluoro-m,m′-dinitrodiphenylsulfone (which forms irreversiblecross-linkages with amino and phenolic groups); dimethyl adipimidate(which is specific for amino groups); phenol-1,4-disulfonylchloride(which reacts principally with amino groups); hexamethylenediisocyanateor diisothiocyanate, or azophenyl-p-diisocyanate (which reactsprincipally with amino groups); glutaraldehyde (which reacts withseveral different side chains) and disdiazobenzidine (which reactsprimarily with tyrosine and histidine).

Cross-linking reagents may be homobifunctional, i.e., having twofunctional groups that undergo the same reaction. A preferredhomobifunctional cross-linking reagent is bismaleimidohexane (“BMH”).BMH contains two maleimide functional groups, which react specificallywith sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7).The two maleimide groups are connected by a hydrocarbon chain.Therefore, BMH is useful for irreversible cross-linking of polypeptidesthat contain cysteine residues.

Cross-linking reagents may also be heterobifunctional.Heterobifunctional cross-linking agents have two different functionalgroups, for example an amine-reactive group and a thiol-reactive group,that will cross-link two proteins having free amines and thiols,respectively. Examples of heterobifunctional cross-linking agents aresuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (“SMCC”),m-maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS”), and succinimide4-(p-maleimidophenyl) butyrate (“SMPB”), an extended chain analog ofMBS. The succinimidyl group of these cross-linkers reacts with a primaryamine, and the thiol-reactive maleimide forms a covalent bond with thethiol of a cysteine residue.

Cross-linking reagents often have low solubility in water. A hydrophilicmoiety, such as a sulfonate group, may be added to the cross-linkingreagent to improve its water solubility. Sulfo-MBS and sulfo-SMCC areexamples of cross-linking reagents modified for water solubility.

Many cross-linking reagents yield a conjugate that is essentiallynon-cleavable under cellular conditions. However, some cross-linkingreagents contain a covalent bond, such as a disulfide, that is cleavableunder cellular conditions. For example, Traut's reagent, dithiobis(succinimidylpropionate) (“DSP”), and N-succinimidyl 3-(2-pyridyldithio)propionate (“SPDP”) are well-known cleavable cross-linkers. The use of acleavable cross-linking reagent permits the cargo moiety to separatefrom the transport polypeptide after delivery into the target cell.Direct disulfide linkage may also be useful.

Numerous cross-linking reagents, including the ones discussed above, arecommercially available. Detailed instructions for their use are readilyavailable from the commercial suppliers. A general reference on proteincross-linking and conjugate preparation is: Wong, CHEMISTRY OF PROTEINCONJUGATION AND CROSS-LINKING, CRC Press (1991).

Chemical cross-linking may include the use of spacer arms. Spacer armsprovide intramolecular flexibility or adjust intramolecular distancesbetween conjugated moieties and thereby may help preserve biologicalactivity. A spacer arm may be in the form of a polypeptide moiety thatincludes spacer amino acids, e.g. proline. Alternatively, a spacer armmay be part of the cross-linking reagent, such as in “long-chain SPDP”(Pierce Chem. Co., Rockford, Ill., cat. No. 21651 H).

The BH3 domain peptides and/or the transduction domain peptides can bepolymers of L-amino acids, D-amino acids, or a combination of both. Forexample, in various embodiments, the peptides are D retro-inversopeptides. The term “retro-inverso isomer” refers to an isomer of alinear peptide in which the direction of the sequence is reversed andthe chirality of each amino acid residue is inverted. See, e.g., Jamesonet al., Nature, 368, 744-746 (1994); Brady et al., Nature, 368, 692-693(1994). The net result of combining D-enantiomers and reverse synthesisis that the positions of carbonyl and amino groups in each amide bondare exchanged, while the position of the side-chain groups at each alphacarbon is preserved. Unless specifically stated otherwise, it ispresumed that any given L-amino acid sequence of the invention may bemade into a D retro-inverso peptide by synthesizing a reverse of thesequence for the corresponding native L-amino acid sequence.

Alternatively, the BH3 domain peptides and/or the transduction domainpeptides are cyclic peptides. Cyclic peptides are prepared by methodsknown in the art. For example, macrocyclization is often accomplished byforming an amide bond between the peptide N- and C-termini, between aside chain and the N- or C-terminus [e.g., with K₃Fe(CN)₆ at pH 8.5](Samson et al., Endocrinology, 137: 5182-5185 (1996)), or between twoamino acid side chains. See, e.g., DeGrado, Adv Protein Chem, 39: 51-124(1988).

BH3 domain peptides and/or the transduction domain peptides are easilyprepared using modern cloning techniques, or may be synthesized by solidstate methods or by site-directed mutagenesis. A BH3 domain peptideand/or the transduction domain peptides may include dominant negativeforms of a polypeptide. In one embodiment, native BH3 domain peptidesand/or transduction domain peptides can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, BH3 domain polypeptidesand/or transduction domain peptides are produced by recombinant DNAtechniques. Alternative to recombinant expression, BH3 domain peptidesand/or transduction domain peptides can be synthesized chemically usingstandard peptide synthesis techniques.

In various embodiments, the BH3 peptide maintains its secondarystructure, e.g. α-helical structure. Methods of helix stabilization areknown in the art.

Preferably, the BH3 peptide is a stable peptide. By “stable “it is meantthat the peptide possess stability sufficient to allow the manufactureand which maintains the integrity of the compound for a sufficientperiod of time to be useful for the purposes detailed herein. Forexample the peptides are covalently stabilized using polar and or labilecrosslinks (Phelan et al. 1997 J. Am. Chem. Soc. 119:455; Leuc et al.2003 Proc. Nat'l. Acad. Sci. USA 100:11273; Bracken et al., 1994 J. Am.Chem. Soc. 116:6432; Yan et al. 2004 Bioorg. Med. Chem. 14:1403).Alternatively, the peptides are stabilized using the metathesis-basedapproach, which employed .alpha., .alpha.-disubstituted non-naturalamino acids containing alkyl tethers (Schafmeister et al., 2000 J. Am.Chem. Soc. 122:5891; Blackwell et al. 1994 Angew Chem. Int. Ed.37:3281). Preferably the peptides are stabilized using hydrocarbonstapling. Stapled peptides are chemically braced or “stapled” peptidesso that their shape, and therefore their activity, is restored and/ormaintained. Stably cross-linking a polypeptide having at least twomodified amino acids (a process termed “hydrocarbon stapling”) can helpto conformationally bestow the native secondary structure of thatpolypeptide. For example, cross-linking a polypeptide predisposed tohave an alpha-helical secondary structure can constrain the polypeptideto its native alpha-helical conformation. The constrained secondarystructure can increase resistance of the polypeptide to proteolyticcleavage and also increase hydrophobicity. Stapled BH3 peptides areproduced for example, as described in WO05044839A2, herein incorporateby reference in its entirety. Alternatively, the BH3 peptides are cyclicpeptides. Cyclic peptides are prepared by methods known in the art. Forexample, macrocyclization is often accomplished by forming an amide bondbetween the peptide N- and C-termini, between a side chain and the N- orC-terminus [e.g., with K₃Fe(CN)₆ at pH 8.5] (Samson et al.,Endocrinology, 137: 5182-5185 (1996)), or between two amino acid sidechains. See, e.g., DeGrado, Adv Protein Chem, 39: 51-124 (1988).

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which the BH3domain peptide is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of BH3peptides and/or transduction domain peptides in which the protein isseparated from cellular components of the cells from which it isisolated or recombinantly produced. In one embodiment, the language“substantially free of cellular material” includes preparations of BH3domain peptides and/or the transduction domain peptides having less thanabout 30% (by dry weight) of non-BH3 domain peptide and/ornon-transduction domain peptides (also referred to herein as a“contaminating protein”), more preferably less than about 20% of non-BH3peptide and/or non-transduction domain peptides, still more preferablyless than about 10% of non-BH3 peptide and/or non-transduction domainpeptides, and most preferably less than about 5% non-BH3 domain peptideand/or non-transduction domain peptides. When the BH3 domain peptideand/or the transduction domain peptides or biologically active portionthereof is recombinantly produced, it is also preferably substantiallyfree of culture medium, i.e., culture medium represents less than about20%, more preferably less than about 10%, and most preferably less thanabout 5% of the volume of the protein preparation. The language“substantially free of chemical precursors or other chemicals” includespreparations of BH3 domain peptides and/or the transduction domainpeptides in which the protein is separated from chemical precursors orother chemicals that are involved in the synthesis of the protein. Inone embodiment, the language “substantially free of chemical precursorsor other chemicals” includes preparations of BH3 domain peptides and/ortransduction domain peptides having less than about 30% (by dry weight)of chemical precursors or non-BH3 domain peptide and/or non-transductiondomain peptides chemicals, more preferably less than about 20% chemicalprecursors or non-BH3 domain peptide and/or non-transduction domainpeptides chemicals, still more preferably less than about 10% chemicalprecursors or non-BH3 domain peptide chemicals, and most preferably lessthan about 5% chemical precursors or non-BH3 domain peptide and/ornon-transduction domain peptides chemicals.

The term “biologically equivalent” is intended to mean that thecompositions of the present invention are capable of demonstrating someor all of the same apoptosis modulating effects, i.e., release ofcytochrome C or BAK oligomerization although not necessarily to the samedegree as the BH3 domain polypeptide deduced from sequences identifiedfrom cDNA libraries of human, rat or mouse origin or produced fromrecombinant expression symptoms.

Percent conservation is calculated from the above alignment by addingthe percentage of identical residues to the percentage of positions atwhich the two residues represent a conservative substitution (defined ashaving a log odds value of greater than or equal to 0.3 in the PAM250residue weight table). Conservation is referenced to sequences asindicated above for identity comparisons. Conservative amino acidchanges satisfying this requirement are: R-K; E-D, Y-F, L-M; V-I, Q-H.

BH3 domain peptides can also include derivatives of BH3 domain peptideswhich are intended to include hybrid and modified forms of BH3 domainpeptides including fusion proteins and BH3 domain peptide fragments andhybrid and modified forms in which certain amino acids have been deletedor replaced and modifications such as where one or more amino acids havebeen changed to a modified amino acid or unusual amino acid andmodifications such as glycosylation so long as the hybrid or modifiedform retains the biological activity of BH3 domain peptides. Byretaining the biological activity, it is meant that cell death isinduced by the BH3 polypeptide, although not necessarily at the samelevel of potency as that of the naturally-occurring BH3 domainpolypeptide identified for human or mouse and that can be produced, forexample, recombinantly. The terms induced and stimulated are usedinterchangeably throughout the specification.

Preferred variants are those that have conservative amino acidsubstitutions made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a BH3domain polypeptide is replaced with another amino acid residue from thesame side chain family. Alternatively, in another embodiment, mutationscan be introduced randomly along all or part of a BH3 coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened to identify mutants that retain activity.

Also included within the meaning of substantially homologous is any BH3domain peptide which may be isolated by virtue of cross-reactivity withantibodies to the BH3 domain peptide described herein or whose encodingnucleotide sequences including genomic DNA, mRNA or cDNA may be isolatedthrough hybridization with the complementary sequence of genomic orsubgenomic nucleotide sequences or cDNA of the BH3 domain peptidesherein or fragments thereof.

Also included in the invention are kits for performing BH3 Profilingusing whole cells. The kit consists of a multi-well plate containingstaining components in a mitochondrial buffer and a tube ofmitochondrial buffer for the suspension and dispensing of cells into theplate for analysis. Each well of the multi-well plate contains a mixtureof JC-1 dye, oligomycin, 2-mercaptoethanol, digitonin, and a peptide orsmall molecule at twice their final concentration, Optionally the plateand suspension buffer tube can be frozen for later use along with thesuspension buffer tube. To use, the plate and buffer tube are thawed andbrought to room temperature. Cells are suspended in buffer, dispensedinto the wells of the plate, and analyzed in a fluorescence plate readerusing the JC-1 red fluorescence at 590 nm with excitation at 545 nm.

The invention will be further illustrated in the following non-limitingexamples.

EXAMPLES Example 1: IBH3: BH3 Profiling by Direct Measurement ofRetained Cytochrome C by FACS

iBH3 adds a key fixation step to prior protocols for BH3 profiling. Thisproduced a better signal, increased sample stability, and improvedstaining to discriminate subsets in complex clinical samples. Primarytissue or cell cultures are dissociated into single cell suspensions,optionally stained for cell surface markers, and suspended in DTEBMitochondria] buffer (BH3 profiling in whole cells by fluorimeter orFACS. Methods. 2013 Apr. 20. Epub ahead of print). The suspended cellsare then added to wells containing DTEB supplemented with digitonin (apermeabilizing agent) and either peptides or small molecules, which canbe prepared and frozen in sample tubes or plates, to allow the moleculesor peptides to access the mitochondria and allow for the free diffusionof cytochrome c out of permeabilized mitochondria and out of the cell.Cells are exposed to peptides/small molecules for period of time beforea short aldehyde fixation followed by neutralization with a Tris/Glycinebuffer. Anti-cytochrome c antibody is then added to each well as aconcentrate with saponin, fetal bovine serum, and bovine serum albuminto stain cytochrome c retained by the cells. Other antibodies tointracellular targets can be added at this time. Cells are analyzed byFACS to provide single cell measurements of cytochrome c afterperturbation with peptides or small molecules to provide diagnosticresponse signatures. (FIG. 1: iBH3 faithfully reproduces the profile ofindividual subpopulations within mixed populations. Samples profiledindividually (unmixed) or as a complex mixture (mixed) produce the sameprofile. This ability to discriminate subpopulations can be applied toany antigen or signal whether intra or extracellular.

This is an improvement over ELISA based BH3 profiling because it cananalyze subpopulations within samples, and it is the only method capableof profiling using both extracellular and intracellular markers.Furthermore, it is capable of performing this analysis in highthroughput format and can be used with pre-made frozen test plateswithout the time sensitivity of live mitochondrial potentialmeasurements using potentiometric dyes.

Example 2: MicroBH3: Single Cell BH3 Profiling by ImmunofluorescenceMicroscopy

MicroBH3 (miBH3) is a BH3 profiling method where the measurement of themitochondrial effect of 9H3 peptides have on individual cells bymicroscopy. To accomplish this, cells are immobilized on polyamine orpoly-lysine coated surfaces and treated with low concentrations ofdigitonin in a mitochondrial buffer to permeabilize the plasma membraneand grant access to the mitochondria without cell disruption. Fixedconcentrations of BH3 peptides or chemical compounds are added for afixed time, generally 45-90 min, before formaldehyde fixation andpermeabilization by methanol and/or Triton X-100(t-Octylphenoxypolyethoxyethanol) for intracellular staining ofcytochrome C and a mitochondrial marker such as MnSOD. Stained cells arecounterstained with nuclear stains such as DAPI, and fluorescent imagesare acquired in nuclear, mitochondrial, and cytochrome c channels.Automated analysis is performed using software such as Cellprofiler tolocate nuclei, define regions adjacent to nuclei that have mitochondria,and then correlate the presence of cytochrome c with the location of themitochondria. Loss of localization indicates a loss of cytochrome c anda reaction to the peptide or compound. This method allows the responseof cells to BH3 peptides or compounds and determine their apoptoticpropensity, or priming, at a single cell level. Previous methods ofanalyzing mitochondrial integrity using potential sensitive fluorescentdyes use intact, not permeabilized, cells and cannot be used with BH3peptides as they are not cell permeant. Permeabilized cells treated withpotential sensitive change shape and are difficult to keep in focus forthe necessary time courses and are sensitive to timing. Fixed cells bythis method can be readily stopped at the fixation step and can beanalyzed weeks after acquisition as well as readily re-analyzed ifneeded.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A method of predicting sensitivity of a cancercell population to a cytotoxic agent, the method comprising: a)providing a single cell suspension of cells from a cancer cellpopulation; b) permeabilizing the cells in the single cell suspension toprovide in the single cell suspension permeabilized cells containingintact mitochondria; c) contacting the permeabilized cells with a BH3domain peptide; d) fixing the permeabilized cells with a fixative; e)staining the fixed permeabilized cells for a molecule from themitochondrial intermembrane space; and f) detecting the staining for themolecule from the mitochondrial intermembrane space to determine anamount of the molecule retained by the permeabilized cells, wherein adecrease in the amount of the molecule retained by the permeabilizedcells in the presence of the BH3 domain peptide compared to an amount ofthe molecule retained by permeabilized cells not contacted with the BH3domain peptide indicates that the cancer cell population is sensitive tothe cytotoxic agent.
 2. The method of claim 1, wherein the fixative isan aldehyde.
 3. The method of claim 1, wherein the fixed cells arestored prior to detecting the staining.
 4. The method of claim 3,wherein the fixed cells are stored for one or more weeks after thefixing.
 5. The method of claim 1, wherein the molecule from themitochondrial intermembrane space is cytochrome c, SMAC/Diablo, Omi,adenylate kinase 2, or apoptosis-inducing factor.
 6. The method of claim1, wherein the staining for the molecule from the mitochondrialintermembrane space is measured by fluorescence-activated cell sorting(FACS).
 7. The method of claim 1, wherein the cells are permeabilizedwith digitonin, methanol, or p-Octylphenoxypolyethoxyethanol.
 8. Themethod of claim 1, wherein the BH3 domain peptide is derived from theBH3 domain of a BH3-interacting domain death agonist (BID), aBcl-2-interacting mediator of cell death (BIM), a Bcl-2-associated deathpromoter (BAD), a Bcl-2-interacting killer (BIK), a Noxa, a p53up-regulated modulator of apoptosis (PUMA), a Bcl-2-modifying factor(BMF), or a harakiri (HRK) polypeptide.
 9. The method of claim 1,wherein the BH3 domain peptide includes a sequence selected from thegroup consisting of SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,and
 13. 10. The method of claim 1, further comprising obtaining thesingle cell suspension of cells from a tissue sample or cell culture.11. The method of claim 1, further comprising staining the cells for acell surface marker, an intracellular marker, or a combination thereofto permit discrimination between cell subpopulations in the cells basedon expression of the cell surface marker, the intracellular marker, orcombination thereof.
 12. The method of claim 11, further comprisingdetecting the staining of the cell surface marker, the intracellularmarker, or the combination thereof, by FACS.
 13. The method of claim 1,wherein the permeabilized cells are contacted with the BH3 domainpeptide for between about 45 minutes and about 90 minutes.
 14. A methodof predicting sensitivity of a cancer cell to a cytotoxic agent, themethod comprising: a) immobilizing the cancer cell on a solid surface;b) permeabilizing the cancer cell to provide a permeabilized cellcontaining intact mitochondria; c) contacting the permeabilized cellwith a BH3 domain peptide; d) fixing the permeabilized cell with afixative; e) staining the fixed permeabilized cell for a molecule fromthe mitochondrial intermembrane space and staining the fixedpermeabilized cell for an intracellular marker; and f) detecting thestaining by microscopy to locate the molecule from the mitochondrialintermembrane space relative to the intracellular marker in the cancercell, wherein loss of localization of the molecule from themitochondrial intermembrane space in the cancer cell indicates that thecancer cell is sensitive to the cytotoxic agent.
 15. The method of claim14, further comprising wherein staining the fixed permeabilized cell forthe intracellular marker comprises staining for one or both of amitochondrial marker or a nuclear marker.
 16. The method of claim 15,wherein the fixed permeabilized cell is stained with DAPI(4′,6-diamidino-2-phenylindole).
 17. The method of claim 15, wherein themitochondrial marker is MnSOD.
 18. The method of claim 14, wherein thesolid surface comprises a polyamine-coated surface or apolylysine-coated surface.
 19. The method of claim 14, wherein themolecule from the mitochondrial intermembrane space is cytochrome c. 20.The method of claim 14, wherein the permeabilized cells are contactedwith the BH3 domain peptide for between about 45 minutes and about 90minutes.